Figure 4

Gating strategy for the tri-colour technique. Plasmodium falciparum NF54 asynchronous culture of ~15% parasitaemia and 2.5% haematocrit was mixed with ~1% human monocyte and stained by the tri-colour technique. Panels A1, B1 and C1 are dot plots of SS Log/FL4-CD45-PerCP, SS Log/FL1-CPO and SS Log/FL3-mitotracker red, respectively, for the control sample containing only monocytes and uRBCs. These were used to set the threshold of positivity for the various dyes. Panel A2 is a dot plot of SS Log/FL4-CD45-PerCP of the test sample to identify and exclude monocytes (CD45 positive cells). Panels B2 (SS Log/FL1-CPO) and C2 (SS Log/FL3-mitotracker red) are dot plots for the same test sample without the exclusion of the monocyte population. The proportion of CD45 negative populations (RBCs) from A2 that were infected with P. falciparum are shown by the SS Log/FL1-CPO dot plot (Panel D). This population includes live and dead (and/or compromised) cells (i.e. total parasitaemia). Live and dead cells from the CPO-positive events were then distinguished in SS Log/FL3-mitotracker red dot plot (Panel E). Quadrants are labeled ‘J’ for CD45-PerCP, ‘G’ for CPO and ‘K’ for mitotracker red plots respectively. The positive (+) and negative (-) signs denote the presence or absence respectively of a given dye signal or cell attribute in the quadrant.